1. Technical Field
This disclosure relates to methods and apparatus for extracting a specimen from a sample substrate at cryogenic temperature for analysis, in particular, methods and apparatus for such extraction inside a charged-particle instrument such as a focused ion-beam microscope (FIB) or scanning electron microscope (SEM).
2. Background Art
While FIB processes have been used in semiconductor and materials science since the 1990's, they are only recently being used by the biological community. Biological samples are generally either embedded in polymeric material such as Epon, or frozen, before sectioning for microscopic imaging. While polymeric-embedded specimens can easily be prepared using the traditional in situ lift-out steps and materials, the lift-out process must be modified to meet requirements related to the frozen specimen.
It is known that traditional gas-assisted ion and electron deposition processes are not viable solutions for bonding the specimen to other objects, such as probe tips or sample holders, at very cold temperatures, since the metallo-organic precursors tend to uncontrollably condense on the specimen in their native precursor form, which results in a suboptimal masking layer for protection of the specimen's top surface during FIB milling. Temperature manipulation can be used, however, to achieve bonding without using gas-assisted deposition. Modifying or maintaining the temperature of frozen biological specimens within the electron microscope is common during manipulation, imaging and analysis of these frozen specimens. To preserve the morphological integrity of a frozen biological specimen during cryo-FIB and cryo-handling methods, however, the specimen temperature in the region of interest should preferably not rise above the vitrification temperature for water, approximately −140° C. Otherwise, ice crystals form within the specimen and damage its structure. Ice-crystal frost must also be avoided for quality imaging and processing of the specimen.
Existing nano-manipulators typically have a moveable probe carrying a fine probe tip. U.S. Pat. No. 7,845,245 (which is not admitted to be prior art by its inclusion in this background section) describes touching a warm probe tip to a vitrified biological specimen, to achieve bonding of the tip to the specimen, based on local induction of a phase change.
This phase change causes the resulting bonding, but also may induce the formation of ice crystals as the sample is warmed above its vitrification temperature at the attachment site. What is needed is a way to create a secure bond of a specimen to a probe tip or other end effector without the risk of devitrification and formation of ice crystals inside the specimen or the formation of frost on the outside of the specimen.